Chen C.C. (ed.) Selected Topics in DNA Repair

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SiDNA and Other Tools for the Indirect Induction of DNA Damage Responses 9

DNA break sensors include the MRN complex (Rupnik et al., 2008), Ku70/80, PARP and RPA.

Break recognition by these sensors leads to the activation of transducer kinases, such as the

PIKKs ATM, ATR and DNA-PK as well as PARP (Iliakis, 2009; Stiff et al., 2004; Ward & Chen,

2001). ATM, DNA-PK and ATR can phosphorylate the serine 139 residue of the histone variant

H2AX (yielding γ-H2AX), at nucleosomes around the lesion. H2AX phosphorylation is

probably the earliest posttranslational modiﬁcation in DDR and may be considered the initial

signal ampliﬁcation step. γ-H2AX formation is followed by binding of the mediator protein

MDC1 (mediator of DNA damage checkpoint 1) to the DSB-ﬂanking chromatin (Jungmichel &

Stucki, 2010). Mediator or adaptor proteins help to transmit, enhance and sustain the signaling

between sensors and transducers, leading to the spread of the repair machinery along the

chromosome. Other mediators include 53BP1 (p53-binding protein 1) and BRCA1 (Li & Zou,

2005; Misteli & Soutoglou, 2009). The recruitment of 53BP1 and BRCA1 to the DSB is indirect,

requiring the activity of the E3 ubiquitin ligases RNF8 (Huen et al., 2007; Kolas et al., 2007) and

RNF168 (Doil et al., 2009; Stewart et al., 2009). ssDNA compartments may be bound by RPA,

which subsequently recruits ATR (Lobrich & Jeggo, 2007) and Rad51. Foci of Rad51 binding

colocalize with Rad52 (Liu & Maizels, 2000), Rad54 (Essers et al., 2002), RPA (Raderschall

et al., 1999), BRCA1 (Scully et al., 1997) and BRCA2 (Chen et al., 1998). Sustained activation

of the transducers results in the transmission of the damage signal to effectors, which relay

the signal to downstream pathways with endpoints in different cellular processes, such as

checkpoint arrest or apoptosis (Kastan & Bartek, 2004) (Figure 3).

Not all the actors in DNA damage signaling and repair form characteristic foci. Unlike ATM,

MDC1, 53BP1, BRCA1/BARD1 and MRN, the central NHEJ proteins Ku70/80 and DNA-PK

do not spread to the adjacent chromatin upon recruitment to the break, presumably because

they are required at low copy number at sites of damage (Bekker-Jensen et al., 2006; Lukas

et al., 2003). The same is true for the effector kinases Chk1 and Chk2, and for p53, which

interact only transiently with damage sites, subsequently diffusing rapidly to relay the signal

to their soluble downstream targets.

One endpoint of the described signaling cascade in response to DNA damage is the activation

of checkpoints to provide the cell with more time for DNA repair. DDR checkpoints have

been identiﬁed at the G1/S and G2/M boundaries, and during S phase and, potentially,

in mitosis (reviewed by Lukas et al. (2004)). After activation, the transducer kinases ATM,

ATR or DNA-PK phosphorylate p53 either directly or via ATM-induced activation of the

effector kinase Chk (checkpoint kinase) 2. Phosphorylated p53 then induces transcription

of the gene encoding the Cdk inhibitor p21, which ultimately prevents transition from G1 to

S-phase. Both Chk 1 and 2 activate the G2/M and intra-S checkpoints (Smith et al., 2010).

It was long thought that ATM principally phosphorylated Chk1 and that ATR preferentially

phosphorylated Chk2. However, this view has been modiﬁed by the discovery of various

crosstalk between these kinases (Bartek & Lukas, 2003). The precise role of DNA-PK in this

regulation remains unclear. PARP may contribute to checkpoint signaling by activation of p53.

p53 exhibits high afﬁnity for automodiﬁed PARP (Malanga et al., 1998) and p53 functions are

impaired in PARP-deﬁcient cells (Wang et al., 1998; Wieler et al., 2003). Furthermore, PARP

activation in response to excessive DNA damage leads to extensive NAD

consumption. The

cellular NAD

depletion can induce cell death through several mechanisms, depending on

the cellular context (reviewed by Rouleau et al. (2010)).

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2.5.2 Complexity of DNA damage response regulation

The outcomes of DNA damage signaling are, literally, a matter of life or death. Depending

on the severity of DNA damage, the cell will either repair the damage to enable it to continue

dividing or enter apoptosis. Complex, redundant signaling pathways converging on several

central node proteins have emerged to ensure that this signaling remains under tight control.

These node proteins must obtain input signals from several sources, in the form of protein

modiﬁcations, before they can relay the signal to downstream effectors. This has a protective

effect, greatly reducing the risk of important cell fate decisions, such as entry into apoptosis,

occurring in response to a single erroneous input signal (Yarosh, 2001). The nodes in DDR

include checkpoint proteins, such as Chk1 and Chk2, which control cell cycling and require

input signals from several sources for full activation (McAdams & Arkin, 1999). Another

prominent example for a node protein in DNA damage signaling is p53 (Kohn, 1999). The p53

protein has 11 sites for phosphorylation and acetylation, and can theoretically assume about

2000 modiﬁcation states, if all the possible independent combinations are taken into account.

Twelve different kinases can phosphorylate p53, and activated p53 can interact with at least

15 downstream proteins (Yarosh, 2001). The kinases that phosphorylate p53 in response to

DNA damage include DNA-PK, ATM and ATR. Full p53 activity requires phosphorylation by

both DNA-PK and ATM, at least (Wang et al., 2000). It is therefore now thought that p53 plays

a key role in determining the degree of damage, through the assessment of input signals, on

which the decision as to whether apoptosis is necessary is based (Kohn, 1999). There is a need

to determine the speciﬁc conditions under which individual PIKKs become activated. Which

(genotoxic) stresses lead to the activation of all transducer kinases? Is it possible to activate a

single PIKK speciﬁcally, without affecting the others, and what are the cellular consequences

of this?

There is also direct interplay between the transducer kinases (Chen et al., 2007). PIKKs

can phosphorylate each other in response to DNA damage, resulting in mutual control of

their activities. In addition to mediating posttranslational modiﬁcations, the kinases seem to

regulate each other, either directly or indirectly (Peng et al., 2005). Studies on mutants and

siRNA experiments have shown that a decrease in the amount of one of these kinases often

leads to a decrease in the amounts of the PIKK sister kinases. The location of the kinases also

seems to play an important role. For ATM, for example, the concentration of multiple copies

in repair foci plays an important role in kinase activation, whereas DNA-PK does not need

such an accumulation of multiple copies for full activation.

Another layer of complexity is added by the partially overlapping substrate speciﬁcities of

the transducers ATM, DNA-PK and ATR (reviewed by Durocher & Jackson (2001) and Yang

et al. (2003)). These transducers signal different types of DNA damage, but it was recently

shown that the PIKK-mediated signaling network is highly extensive, with hundreds of

phosphorylation events at ATR, ATM and DNA-PK consensus target sites induced by IR

(Matsuoka et al., 2007). As discussed above, the plethora of types of damage induced by

IR results in the activation of all three PIKKs. It remains to be determined which substrates

are speciﬁc or overlapping for which transducer kinases in this long list of potential PIKK

targets. Further insight into the contributions of individual repair signaling pathways has

been provided by studies of the responses induced by damage signals in the absence of

chromatin damage. This aspect will be discussed below.

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3. Methods and mechanisms for inducing a damage response in the absence of

chromatin damage

The induction of DNA damage repair pathways by exogenous DNA was ﬁrst reported in

bacteria, into which UV-irradiated lambda bacteriophages (D’Ari & Huisman, 1982; George

et al., 1974) or plasmids (Bailone et al., 1984) were introduced. This indirect response is

controlled by activation at the sites of exogenous DNA damage of the RecA protein, the

key enzyme of the bacterial damage signaling response known as the “SOS response” (see

Schlacher & Goodman (2007)). No such mechanism was initially found in mammals, despite

a few publications reporting that UV-irradiated H-1 parvovirus or SV40 simian virus induced

an “SOS-like” repair pathway in infected mammalian cells (Cornelis et al., 1982). Elucidation

of the mechanisms underlying the response to damaged DNA or RNA in the cell took much

longer in mammals. We review here the mammalian cell response to oligonucleotides, viral

and immunostimulatory DNA and the insight into DDR gained from the analysis of artiﬁcial

repair foci.

3.1 Cellular response to DNA oligonucleotides

In bacteria, ssDNA has been identiﬁed as the signal triggering the bacterial SOS repair

response. The RecA protein (Rad51 in humans) is directly stimulated by ssDNA, inactivating

the LexA repressor and triggering the repair response (Craig & Roberts, 1981). It has been

suggested that ssDNA (at SSBs, stalled replication forks or resected DSBs) acts also as the

major stimulatory signal for DNA damage responses in eukaryotic cells (Li & Deshaies, 1993;

Nur et al., 2003). Several studies based on transfection or the microinjection of synthetic

DNA oligonucleotides have analyzed the response of the cell to ssDNA. Studies by Nur et al.

(2003) have shown that ssDNA acts upstream from ATM/p53 in DNA damage signaling. The

transfection of cells with short (as few as 5 bases) ssDNA molecules with random sequences

induced ATM activation and apoptosis, whereas very short (8 bp) dsDNA molecules did not

(Nur et al., 2003). The induction of apoptosis by ssDNA is consistent with earlier studies, in

which transfection with randomly fragmented DNA (Schiavone et al., 2000) or the nuclear

injection of linearized plasmid DNA, circular DNA containing a gap, or single-stranded

circular phagemids induced cell cycle arrest or apoptosis (Huang et al., 1996). The activation

of ATM by fragmented DNA requires the MRN-assisted assembly of short, linear ssDNA

fragments into high-molecular weight complexes, as shown by experiments in Xenopus laevis

extracts (Costanzo et al., 2004).

So, if ssDNA can activate ATM directly, how is ATM activated in response to DNA

double-strand breaks? When DNA double-strand breaks are sensed by the MRN complex,

MRN partially unwinds the ends to expose ssDNA (Lee & Paull, 2005). It has been shown in

Xenopus laevis egg extracts that 70 bp synthetic double-stranded molecules are rapidly resected

in an MRN-dependent manner to generate ssDNA oligonucleotides, which activate ATM

(Jazayeri et al., 2008). Consistent with these ﬁndings, the injection of small synthetic ssDNA

oligomers into undamaged cells also induces ATM activation, and the elimination of ssDNA

oligomers results in the rapid extinction of ATM activity. In summary, the results obtained

from experiments with single-stranded or long double-stranded DNA fragments suggest that

short ssDNA molecules are the essential signal for the induction of ATM-dependent cell cycle

arrest and apoptosis.

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The cellular response to single-stranded oligonucleotides may also have consequences for

targeted gene repair with synthetic oligonucleotides. In gene targeting processes, ssDNA

molecules are transported to the nucleus, where they align with their complementary

sequence in the target gene, facilitating nucleotide exchange (Brachman & Kmiec, 2002).

However, the introduction of large amounts of oligonucleotides into cells may induce cell

cycle arrest and stalled DNA replication in the corrected cells, due to the ATM activation

by the mechanism described above. Ferrara & Kmiec (2006) showed that the transfection of

human colorectal cancer cells with 47-mer single-stranded oligonucleotides mostly activated

Chk1 and Chk2 in corrected cells. As a consequence, uncorrected cells may outgrow corrected

cells. The uncorrected cells probably contain fewer oligonucleotides, too few indeed to

generate a local ssDNA concentration high enough to activate ATM. This may account for

previous reports of the decline of corrected populations over time (Igoucheva et al., 2004).

3.2 Synthetic interstrand crosslinks

The cytotoxic activity of many chemotherapeutic agents, including cisplatin, nitrogen

mustards and mitomycin C, is due to the induction of interstrand crosslinks (ICLs) in DNA.

These lesions block the strand separation necessary for essential DNA functions, such as

transcription and replication. ICLs affect both strands of the chromosome, and their repair is

particularly complex. It involves factors from multiple repair pathways and the mechanisms

may differ at different stages of the cell cycle. Nucleotide-excision repair, homologous

recombination repair, the Fanconi anemia repair pathway, MMR and translesion synthesis

have all been shown to participate in ICL repair.

Oligonucleotides containing synthetic ICLs are a valuable tool for the study of ICL repair

(Guainazzi & Scharer, 2010). The advantage of such molecules over cell treatment with a

cross-linking agent is that it is possible to study the components involved in the repair of

speciﬁc interstrand crosslink products one at a time. This has mostly involved the ligation

of the synthetic ICL-containing DNA fragments into plasmids, followed by analysis of their

repair in cell-free extracts or cells (Orelli et al., 2010; Raschle et al., 2008; Wang et al., 2001).

The repair of the speciﬁc ICL can be analyzed with reporter genes or by enzymatic digestion

followed by Southern blotting. Studies with synthetic DNA ICLs have contributed to our

understanding of the mechanism of replication-coupled DNA ICL repair (Wang et al., 2001)

and the role of the Fanconi anemia pathway in this repair process (Knipscheer et al., 2009).

3.3 Viral infection

The highly organized cellular response to DNA damage may be disorganized or hijacked

during viral infection (Lilley et al., 2007; Weitzman et al., 2004). Viruses often produce large

amounts of exogenous DNA during infection, and generate proteins that interfere with DNA

repair pathways and cell cycle checkpoints. Recent studies have suggested that the cellular

DNA repair machinery can recognize viral nucleic acids as damage (Lilley et al., 2007). In

some cases, the host DNA repair response is inactivated by viruses, whereas in others, the

maintenance of a functional host DNA repair machinery seems to increase viral replication.

Studies of the interaction of viruses with the host cell DNA repair machinery have improved

our understanding of normal cellular DNA repair functions, and viruses may be useful model

systems for studying certain aspects of DNA repair (Weitzman & Ornelles, 2005). In this

section, we provide an overview of the ways in which viral infection can activate the DDR in

cells, thereby focusing on the response to viral genomic material. For a detailed description

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of the ways in which viral proteins can manipulate the host DNA repair machinery and cell

cycle checkpoints, we refer the reader to Chaurushiya & Weitzman (2009), Davy & Doorbar

(2007) and Weitzman et al. (2004).

3.3.1 DDR induction by DNA viruses

Adenoviruses (Ad), which belong to the parvovirus family, are probably the most thoroughly

investigated DNA viruses. Their genome consists of a linear, 36 kb dsDNA molecule, with

inverted terminal repeats (ITRs) at each end and origins for DNA replication (Weitzman

& Ornelles, 2005). Viral proteins synthesized before viral DNA replication prevent the

recognition of the Ad genome by host DNA repair factors. This may involve the targeting,

by these proteins, of cellular damage sensors and repair effectors, such as the MRN complex

or DNA ligase IV, for degradation and relocalization (Carson et al., 2003; Stracker et al.,

2002). Infection with Ad lacking the proteins required for blockade of the host DNA repair

machinery results in partial DDR activation. DNA damage mediator proteins accumulate

at sites of viral replication (Stracker et al., 2002) and γ-H2AX is formed at the periphery of

viral centers, in a process dependent on host cell PIKK activity (Carson et al., 2003). Despite

the interference of viral proteins with the cellular DNA repair system during infection with

wild-type Ad, the cellular DDR does not seem to be completely abolished. The foreign

DNA induces H2AX phosphorylation during later stages of infection, after the onset of

viral DNA replication (Nichols et al., 2009). H2AX phosphorylation follows a pan-nuclear

pattern, suggesting that all the H2AX on the host chromatin is phosphorylated by ectopic

kinase activation, contrasting with the localized γ-H2AX formation observed in response

to DSBs in the chromatin. Viral replication seems to be required for this phosphorylation,

because infection with a nonreplicating virus does not induce γ-H2AX (Nichols et al., 2009)

production. ATR may be the principal kinase phosphorylating H2AX in these conditions,

although ATM and DNA-PK also seem to be involved (Nichols et al., 2009).

Pan-nuclear H2AX phosphorylation has also been observed in cells infected with the

adeno-associated virus (AAV) (Collaco et al., 2009; Fragkos et al., 2008; Schwartz et al.,

2009). The AAV genome, like that of Ad, consists of an ssDNA molecule with ITRs at both

ends, resulting in the formation of double-hairpin structures (Brown, 2010). AAV infection

requires helper functions, which may be supplied by Ad or other viruses (Geoffroy & Salvetti,

2005), and components of the host cell DNA replication machinery (Nash et al., 2009). Viral

replication takes place in the nucleus, where cellular proteins, including RPA, colocalize

with viral proteins in replication centers (Stracker et al., 2004). AAV replication in the

presence of minimal Ad helper proteins induces a robust DDR-like response. This response is

independent of the MRN complex and seems to be mediated principally by DNA-PKcs and,

to a lesser extent, by ATM (Collaco et al., 2009; Schwartz et al., 2009). The response involves

the accumulation of DNA-PK in compartments in which viral replication is occurring, and the

pan-nuclear phosphorylation, not only of H2AX, but also of Smc1 and ATM (Schwartz et al.,

2009). In another study, the phosphorylation of RPA, Nbs1 and Chk1/2 was observed, but the

phosphorylation pattern was not investigated (Collaco et al., 2009).

In addition to the DNA-PK-dependent DDR induced by AAV replication, studies on

recombinant AAV (rAAV) vectors (Carter, 2004), have demonstrated the existence of a

requirement for DNA-PKcs and Ku70/80 for viral DNA replication (Choi et al., 2010).

The inactivation of DNA-PK by a DNA-PK inhibitor or siRNA signiﬁcantly decreases the

replication of rAAV, and any rAAV DNA that is replicated forms head-to-head or tail-to-tail

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junctions. Moreover, AAV-ITRs interact directly with Ku proteins, suggesting that viral

DNA is recognized directly by cellular NHEJ factors (Choi et al., 2010). Furthermore,

DNA-PKcs and Artemis have been shown to open the ITR hairpin loops of rAAV in vivo,

in a tissue-dependent manner. In the absence of either of these factors, double-stranded linear

rAAV genomes capped with covalently closed hairpins at their termini accumulate in cells

(Inagaki et al., 2007), conﬁrming the importance of host cell NHEJ proteins for viral DNA

end processing. By contrast to the “hyperactivation” of DNA-PK observed in response to

replicating AAV, the DNA of UV-inactivated AAV particles activates a DDR involving ATM

and ATR, leading to the inhibition of cell cycle progression (Jurvansuu et al., 2005). It has been

suggested that the UV-treated DNA mimics stalled replication forks (Jurvansuu et al., 2005),

whereas DNA-PK activation is consistent with the presence of DSBs as intermediate products

during AAV replication.

DNA-PKcs and Ku70/80 as well as other cellular repair or replication factors including

Topoisomerases I and II, MSH2-MSH6, RecQL, PARP and scaffold attachment factor A

(SAF-A) are involved in Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication

(Wang et al., 2008). KSHV is a large dsDNA virus and the etiologic agent of several

AIDS-associated cancers, including Kaposi’s sarcoma (Antman & Chang, 2000). The lytic

replication of KSHV and the continuous primary infection of fresh cells are responsible

for viral tumorigenicity and pathogenesis, by contrast to what has been reported for other

oncogenic viruses (Grundhoff & Ganem, 2004). The proteins listed above bind to KSHV DNA

fragments and accumulate in viral replication compartments in the nucleus, suggesting a

possible role for these host replication and repair proteins in the viral lytic replication process

(Wang et al., 2008).

In the case of infection with herpes simplex virus type 1 (HSV-1), which contains a linear

dsDNA molecule, a DNA damage signaling is induced which depends principally on ATR

(Lilley et al., 2005; Wilkinson & Weller, 2006). Several members of the cellular DNA

damage-sensing machinery, including RPA, RAD51 and Nbs1, are activated and redistributed

during viral DNA replication, indicating that infection induces the host response to DNA

damage. H2AX is phosphorylated but the γ-H2AX signal is marginalized to the periphery of

viral replication centers (Wilkinson & Weller, 2006). HSV-1 sequesters hyperphosphorylated

RPA away from viral replication compartments, thus preventing a normal ATR-signaling

response. The partial and mislocalized activation of the cellular DDR leads ultimately to its

disorganization.

ATR is also activated during viral replication of the dsDNA virus human cytomegalovirus

(HCMV), leading to an induction of γ-H2AX (Luo et al., 2007). Contrary to what has

been observed for HSV-1, γ-H2AX co-localizes with viral replication compartments during

late-stage infections with HCMV. Only a subset of proteins in addition to γ-H2AX was

speciﬁcally sequestered in viral replication centers, with other proteins excluded from these

centers, impeding both the efﬁcient repair of viral DNA and checkpoint activation (Luo et al.,

2007).

ATM-dependent checkpoint signaling takes place during induction of Epstein Barr virus

lytic replication (Kudoh et al., 2005) or polyomavirus replication (Dahl et al., 2005). This

is accompanied by the phosphorylation of a number of DNA damage markers including

H2AX. The cellular ATM activation in response to both viruses leads to a prolonged S-phase

advantageous for viral lytic replication. However, both viruses have developed efﬁcient

strategies to block ATM-induced p53 downstream signaling that would eventually lead to

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apoptosis (Dahl et al., 2005; Kudoh et al., 2005). By contrast, the autonomous minute virus

of canines (MVC), a member of the parvovirus family, induces apoptosis and G(2)/M-phase

arrest in infected canine cells (Chen et al., 2010). This seems to be the result of the induction

of an ATM/p53-mediated DDR (Luo et al., 2011). Infected cells display phosphorylation of

H2AX, RPA, ATM and ATR. The inhibition or knockdown of ATM decreased cell death and

reduced MVC DNA replication.

In summary, studies of the response of the cell to viral genomic material have demonstrated

that the activation of a damage response involving ATM (e.g. in the case of MVC) or

ATR (UV-inactivated rAAV) leads to cell cycle arrest or p53-dependent cell death, unless

downstream effectors are inactivated or sequestered by viral proteins (HSV-1, HCMV). By

contrast, DNA-PK activation (Ad, AAV) leads to pan-nuclear H2AX phosphorylation, which

does not seem to be detrimental to the cell. These ﬁndings have possible consequences for the

choice of viral vectors for gene targeting. It remains to be determined whether the “ectopic”

phosphorylation of DNA-PK downstream targets affects the stability of the host cell genome

or impairs DNA repair in infected cells. It has also been shown that the speciﬁc recruitment of

a subset of cellular DNA repair factors is essential for viral DNA replication, suggesting that

inhibitors targeting these proteins may have antiviral activity.

3.3.2 DNA damage signals induced by retroviruses

Interaction with the cellular DNA damage sensing machinery is not limited to DNA viruses.

Retroviruses, which contain a single-stranded RNA genome, use a viral-encoded reverse

transcriptase to generate a cDNA that is then integrated into the host genome. The

preintegration complex contains the cDNA and the viral-encoded integrase enzyme. This

enzyme then mediates strand transfer to integrate the viral DNA into the host DNA in a non

site-speciﬁc fashion (Weitzman et al., 2004). The interaction of retroviruses with the cellular

DNA repair machinery has been studied in detail (Lilley et al., 2007; Sakurai et al., 2009;

Skalka & Katz, 2005). The NHEJ pathway has been implicated in the sensing and processing

of the linear cDNA, although the exact step of the viral life-cycle affected by the cellular DNA

repair machinery remains unclear Ariumi et al. (2005). It is possible that the viral cDNA

acts directly as a substrate for NHEJ, which circularizes the DNA by end ligation (Li et al.,

2001). The circularization of unintegrated DNA in this context may protect the viral DNA from

degradation and the cell from apoptosis (Kilzer et al., 2003). Consistent with this hypothesis,

the infection with retrovirus of cells lacking NHEJ factors results in apoptosis, suggesting that

DNA-PKcs and Ku70/80 may protect against the cellular toxicity of high levels of retroviral

cDNA (Li et al., 2001).

Another interaction of retroviruses with the DNA repair machinery is constituted by the

role of cellular repair and damage signaling factors in the integration of viral DNA into

the host genome (Daniel et al., 1999). During retroviral integration, DSBs are created as an

intermediate that may be detected as DNA damage by the host cell (Sakurai et al., 2009).

Transient phosphorylation of histone H2AX occurs at retroviral integration sites, and it has

been suggested that the completion of the integration process is dependent on DNA-PK

(Daniel et al., 1999; 2004). DNA-PK-deﬁcient mouse scid cells infected with three different

retroviruses display much lower levels of DNA integration than wild-type cells and die

by apoptosis. Furthermore, it has been reported that DNA-PKcs is required for efﬁcient

transduction by retroviral vectors (Daniel et al., 1999). Other PIKKs have since been shown to

be activated after HIV-1 infection and to play a potential role in provirus integration. The use

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of caffeine or a small-molecule inhibitor to block the ATM activity results in the inhibition of

HIV transduction and replication (Daniel et al., 2005; Lau et al., 2005).

DNA-PK and ATM also seem to be important for correct end processing at the junctions

between HIV-1 provirus and host DNA (Sakurai et al., 2009). Large numbers of abnormal

junctions are observed in Mre11- and DNA-PKcs-deﬁcient cells, and Artemis-deﬁcient cells

also display such abnormalities, suggesting a role for NHEJ processing factors in correct viral

integration. Another study has reported a role for Ku70/80 in the targeting of retroviral

elements to chromatin domains prone to gene silencing. Higher levels of viral DNA

expression are observed in the absence of Ku70/80, with no effect on viral transduction

(Masson et al., 2007).

The importance of cellular components of the DDR for the replication of some viruses could be

exploited for therapeutic purposes. For instance, a small-molecule ATM-inhibitor displayed

antiviral activity in a proof-of-concept study with HIV-1, in which ATM deﬁciency sensitized

cells to retrovirus-induced cell death (Lau et al., 2005).

3.4 Tethering of repair factors

Recent studies in yeast and mammals have demonstrated that it is not the DNA lesion that

triggers the DDR, leading to the activation of transducer kinases, but the local concentration

of signaling factors at repair foci. Soutoglou & Misteli (2008) demonstrated that a DDR can

be triggered in the absence of DNA lesions. The artiﬁcial localized tethering of repair factors

was found to be sufﬁcient to trigger a full DDR response, including cell-cycle arrest, in the

absence of DNA breaks. Tethering was achieved by fusing repair proteins to the Escherichia

coli lac repressor and expressing the resulting constructs in mouse NIH-3T3 cells containing

multiple lacR binding sites, stably integrated into the genome (Soutoglou & Misteli, 2008).

The immobilization of Nbs1, Mre11 or MDC1 alone led to DDR activation. In addition, the

tethering of ATM was sufﬁcient to induce ATM kinase activity and autophosphorylation.

Consistent with its function downstream from the MRN complex, the induction of ATM

activity was not affected by an absence of Mre11 or Nbs1. As similar results were obtained

in analogous experiments in Saccharomyces cerevisiae (Bonilla et al., 2008), the concentration of

key factors on the chromatin may be considered a universal characteristic of DDR initiation

and ampliﬁcation in eukaryotes (Misteli & Soutoglou, 2009). The tethering experiments may

be seen as complementary to the experiments with siDNA described below, as the DNA

break-mimicking DNA molecules are (i) too short for the formation of repair foci and (ii) not

in a chromatin context.

3.5 DDR induction by cofactors, oxidative stress or changes in chromatin state

ATM, and consequently the DDR, can be activated in the absence of DNA breaks, by changes

in chromatin structure. Bakkenist & Kastan (2003) showed that ATM is rapidly activated by

the exposure of cells to chromatin-active agents, such as mild hypotonic buffers, or treatment

with chloroquine. Furthermore, histone deacetylase (HDAC) inhibitors, such as TSA, which

induces the global decondensation of chromatin, can also induce ATM activity (Jang et al.,

2010; Mazumdar et al., 2006). Consistent with its role as a sensor of chromatin structure, ATM

can interact with the chromatin via the nucleosome-binding protein HMGN1 before DNA

damage occurs. The loss of this interaction in the absence of HMGN1 compromises ATM

activation in response to IR (Kim et al., 2009).

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ATM-dependent phosphorylation of H2AX during M-phase has been reported to occur in

the absence of DNA damage (Ichijima et al., 2005; Mazumdar et al., 2006). The high degree

of compaction of mitotic chromosomes may be the signal responsible for activating DNA

damage signaling kinases in the absence of apparent DNA lesions (Ichijima et al., 2005).

Another recent study showed that DNA-damage signaling pathways and ATM could be

activated directly by hypoxia, apparently in the absence of lesions (Bencokova et al., 2009).

The direct mechanism involved is unknown, but the hypoxia-induced activation of ATM is

independent of the MRN complex, and active, autophosphorylated ATM has a pan-nuclear

rather than focal distribution. The activation of ATM signaling in response to hypoxia may

account for the cessation of replication when oxygen levels are low.

ATM is a also a direct sensor of oxidative stress, in the absence of DNA lesions. Oxidation

by ROS results in the formation of an active, disulﬁde-cross-linked, ATM dimer (Guo et al.,

2010). The activation of ATM in response to oxidative stress is independent of the MRN

complex and leads to the phosphorylation of DDR effectors including p53 and Chk2, but not

the chromatin-associated proteins H2AX or Kap1. This conﬁrms that ATM activation through

oxidation may occur without the involvement of the DNA damage recognition machinery.

Consistent with ATM activation, the transducer kinase ATR can also be activated in the

absence of DNA damage. Toledo et al. (2008) reported the ectopic activation of ATR following

overproduction of a fragment of TopBP1 containing a domain known to stimulate ATR kinase

activity (Kumagai et al., 2006). ATR activation was sufﬁcient to drive cell cycle arrest and

senescence.

3.6 Systemic response to foreign DNA

DNA fragments trigger a DDR at the cellular level, but the detection of foreign DNA by

speciﬁc sensors can trigger an innate immune response at the systemic level (for review see

Vilaysane & Muruve (2009), Yanai et al. (2009) and Rathinam & Fitzgerald (2011)). The innate

immune system is an integral part of the host response to viral and bacterial intrusion. Its

activation leads to diverse cellular responses, including the induction of interferon regulatory

factors (IRF) 3 and 7, which regulate the production of type I interferon. Furthermore, NFκB

is induced, and regulates the expression of pro-inﬂammatory cytokines (Vilaysane & Muruve,

2009). We provide here a brief overview of the principal sensors of foreign DNA found in

humans.

Bacterial DNA differs from mammalian DNA principally in terms of its high CpG

dinucleotide content. Furthermore, most of the small number of CpG dinucleotides present

in mammalian DNA are methylated, whereas the CpG dinucleotides in bacterial DNA are

generally unmethylated (Hemmi et al., 2000). The activation of innate immune responses

by nucleic acids is mediated by transmembrane Toll-like receptors (TLRs) and cytosolic

receptors. The best understood sensor of microbial DNA is TLR9, which is found principally

in plasmacytoid dendritic cells (pDCs) in humans (Hemmi et al., 2000; Wagner, 2004). The

endosomal uptake of CpG DNA activates TLR9, which binds MyD88, IRAK4 and IRAK1 that

are required for the activation of NFκB and the induction of type I interferons via IRF7 (Akira

et al., 2006; Vilaysane & Muruve, 2009). Several studies have also shown that, in addition to

responding to bacterial DNA, TLR9 plays an important role in host defense against viruses,

including DNA viruses such as herpes simplex virus and murine cytomegalovirus (Delale

et al., 2005; Lund et al., 2003).

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18 Will-be-set-by-IN-TECH

In addition to the membrane-bound TLR9, cytosolic DNA sensors have recently been

identiﬁed (Yanai et al., 2009). These sensors include TREX1, DNA-dependent activator of

interferon regulatory factors (DAI) and a DNA-sensing inﬂammasome containing absent

in melanoma 2 (AIM2). This protein belongs to the HIN-200 family and is a cytoplasmic

sensor mediating caspase 1 activation in response to cytoplasmic dsDNA (Burckstummer

et al., 2009; Fernandes-Alnemri et al., 2009; Hornung et al., 2009). Another cytosolic DNA

sensor and activator of innate immune responses, DAI (also known as DLM-1 and Z-DNA

binding protein 1), has also recently been identiﬁed (Takaoka et al., 2007). DAI binds dsDNA,

leading to its association with the IRF3 transcription factor and the TBK1 serine/threonine

kinase, resulting in a signaling cascade that culminates in NFκB activation. The activation of

interferon responses is restricted by the length of the DNA, with little activation observed in

response to DNA molecules of less than 100 bp in length, suggesting that DAI activation

requires the formation of a multimeric complex over long stretches of DNA (Takaoka &

Taniguchi, 2008).

Several studies have identiﬁed the 3’

→5’ DNA exonuclease DNaseIII/TREX1 as associated

with autoimmune and inﬂammatory diseases in humans (Crow et al., 2006; Lee-Kirsch

et al., 2007). TREX1 is a regulator of DNA homeostasis in the cell and has been reported

to counteract the activation of ATM by small ssDNA fragments (section 3.1). Indeed, in

the absence of TREX1, 60-65 bp ssDNA polynucleotides accumulate, leading to chronic

ATM-dependent DNA damage checkpoint signaling (Yang et al., 2007). Thus, TREX1 is not

itself a DNA sensor. Instead, it regulates the accumulation of the ssDNA that can trigger an

innate immune response. In addition to linking a DDR factor to the immune response, these

ﬁndings demonstrate that immune responses may be triggered not only by foreign DNA, but

also by self-derived DNA.

The most prominent protein involved in both the DDR and the immune response, in

addition to TREX1, is DNA-PKcs. In addition to its long known role in V(D)J recombination

in developing lymphocytes (Jeggo et al., 1995), DNA-PKcs has also been reported to

phosphorylate IFN regulatory factor-3 (IRF-3) directly (Karpova et al., 2002). IRF-3 plays a

key role in the host response to viral infection, and its phosphorylation by DNA-PK after viral

infection results in its nuclear retention and delayed proteolysis. Another study has suggested

that DNA-PKcs mediates Akt activation in response to CpG-DNA in bone marrow-derived

macrophages (BMDMs) (Dragoi et al., 2005). In BMDMs, DNA-PKcs associates with Akt

upon CpG-DNA stimulation, thereby triggering the nuclear translocation of Akt. It has been

suggested that TLR9 is involved in this pathway, but its possible role remains a matter of

debate (Dragoi et al., 2005; Sester et al., 2006). The CpG-DNA/DNAPKcs/Akt pathway may

account for the induction, by CpG-DNA and synthetic CpG oligonucleotides, of prosurvival

signals delivered to the immune system (Dragoi et al., 2005; Park et al., 2002).

4. Signal interfering DNA (siDNA)

As summarized in Figures 1 and 2, difﬁculties attributing the activation of a speciﬁc

transducer to a speciﬁc type of damage in vivo result principally from the multiple type of

damage induced by a single treatment, through the mechanism of action of the treatment and

metabolism of the initial damage through replication and repair. Moreover, the large DNA

molecules sometimes used to induce a damage response (see section 3.1) probably contain

sequences recognized by speciﬁc proteins that may interfere with DNA damage recognition

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Selected Topics in DNA Repair